More specifically, this invention relates to the biosensors that are used to measure the amount of analytes in bodily fluids, particularly in measuring glucose in samples of whole blood. Optical methods are often used for making such measurements, but the present invention relates to improvements in electrochemical biosensors.
Although the methods of the invention to be described herein can be applied to measuring other analytes, measuring glucose in whole blood samples is of particular interest. The invention also relates to an electrochemical instrument in which a constant or varying potential is applied to electrodes in contact with a blood sample and the resulting current is measured after a short period of time. The measured electrical current is correlated with the amount of the analyte in the sample. Such instruments are referred to as amperometric.
Glucose biosensors used in amperometric instruments may employ a number of reagent systems that react enzymes with the glucose in the sample and produce a measurable electrical current by oxidation of a redox compound, referred to as a mediator in the following general sequence of steps:Glucose+Eoxid→Ered+Oxidized Glucose (Gluconolactone)Ered+n Medoxid→n Medred+Eoxid n Medred→Medoxid+n e−
Where Eoxid and Ered are oxidized and reduced forms of the redox center of the enzyme and Medoxid and Medred are the oxidized and reduced forms of the mediator. Glucose oxidase has been used as the enzyme in electrochemical biosensors, but more recently, glucose dehydrogenase has been introduced. These enzymes are used with a co-enzyme or co-factor, such as NAD, FAD, and PQQ. The mediator may be ferricyanide or a tetrazolium salt, among others familiar to those skilled in the art.
Glucose dehydrogenase (GDH), its co-factor, and the mediator are combined in a formulation that is applied to a pair of electrodes, described as the working and counter electrodes. When a potential is applied across the electrodes, the enzyme/co-factor oxidizes the glucose (the analyte) and the mediator is reduced as it reoxidizes the enzyme. The reduced mediator migrates to the working electrode where it is reoxidized, and in the process releases electrons that move to the counter electrode and establish an electrical current that is proportional to the amount of the glucose present in the sample.
Since a new sensor is used each time a patient tests the amount of glucose in their blood, reagent formulations should provide consistent performance from one biosensor to the next. Clearly, it is important that the results are reliable, because the users will adjust their diet or medication in response to the results of their tests. Therefore, among other requirements, the enzyme should maintain its activity throughout its useful shelf life. The present invention relates in particular to limiting or preventing the loss of activity of an enzyme/co-factor system [i.e., glucose dehydrogenase-pyrrolo-quinoline quinone (GDH-PQQ)] used in electrochemical biosensors.
One method of maintaining activity of GDH-PQQ is suggested in U.S. Pat. No. 6,656,702, which teaches the addition of sugars to the reagent formulations, particularly trehalose with GDH-PQQ. In one example a hydrophilic polymer, carboxymethyl cellulose, is deposited on the electrodes and dried. Then, the reagent mixture, including GDH-PQQ, trehalose, and potassium ferricyanide as the electron acceptor (mediator), was deposited by “dropping” on the dried carboxymethyl cellulose layer and then dried to complete the reagent layer on the electrodes. The patent also suggests that a hydrophilic polymer could also be added to the layer that contains the reagents. It is believed that the '702 patent by referring to “dropping” of the reagent layers means that they were deposited by dispensing reagent droplets into a well surrounding the exposed electrodes.
Another patent (U.S. Pat. No. 6,270,637) describes a formulation for electrochemical biosensors that includes GDH-PQQ and also includes hydroxyethyl cellulose. The patent stresses the value of including polyethylene oxide having a molecular weight of 100 to 900 kilodaltons (kDa). The method of applying their formulation on the electrodes is not provided in detail, but it is believed to be done by dispensing reagent with a pump system.
Screen printing of reagent layers was used in the compositions and biosensors described in U.S. Pat. Nos. 5,708,247; 5,951,836; and 6,241,862. Glucose oxidase was used as the enzyme and the reagent compositions also included hydroxyethyl cellulose and a treated silica that was selected to have a balance of hydrophobicity and hydrophilicity, said to form two-dimensional networks that exclude red blood cells.
Reagent formulations may be deposited by various methods, including impregnation, stripe coating, ink-jet printing, or micro-deposition with a syringe pump that may include the addition of a micro-solenoid valve drop ejection device. Screen printing is a method of particular interest since it is efficient and well adapted to large scale production of biosensors. It requires that the formulation (i.e., the ink) applied to the electrodes has certain physical properties to be successfully applied. In particular, the inks applied by screen-printing should have the following properties: adhesion to the substrate, cohesion, thixotropy (shear thinning), and optimized rheology for viscosity and flow.
The present inventors found that a composition, which was used for screen printing when glucose oxidase was the enzyme, could not be used when the enzyme was changed to GDH-PQQ, because the new enzyme lost activity rapidly. After investigation, it was determined that certain of the components used to provide the necessary physical properties for screen printing caused the loss of enzyme activity. Consequently, it was necessary to discover components that would not cause premature deactivation of the GDH-PQQ, but also meet the requirements for screen printing. Those reagent formulations are described below.